Artificially culturing microorganisms in a specific environment requires various nutrient components to be added to a culture medium, and the required nutrient components vary depending on the types of microorganisms to be cultured. To meet the nutrient requirements, generally various culture media containing nutrient components of minerals, amino acids, vitamins, peptones, corn steep liquor, yeast extract, and the like have been developed and used. A culture medium for microorganisms is largely classified into a synthetic medium only containing chemically defined nutrient components such as minerals, amino acids, vitamins, and the like, and a complex medium containing chemically undefined nutrient components such as peptones, corn steep liquor, yeast extract, and the like.
To develop a synthetic medium, identification of all the nutrient components by a corresponding type of microorganism is required, since required nutrient components vary depending on the types of microorganisms as described above. Generally, in order to identify all the required nutrient components, all amino acids and vitamins are added to a culture medium containing minerals, so as to culture a given microorganism on the culture medium, and to check whether the microorganism grows. When growth of the microorganism is observed, after preparing a fresh culture medium by removing one of the nutrient components added to the culture medium, the microorganism is cultured on the culture medium from which a specific nutrient component is removed, and then, a check is performed to determine whether the microorganism grows. When growth of the microorganism is observed, another fresh culture medium is prepared by removing another nutrient component from among the nutrient components added to the initial culture medium. Conversely, when growth of the microorganism is not observed, the removed nutrient component is determined to be an essential nutrient component that is to be added during preparation of a synthetic medium, and afterwards, is necessarily added when preparing a culture medium. Recently, a synthetic medium has been developed by repeatedly performing this method of identifying all the nutrient components necessary for microorganism cultivation (Zhang et al., App. Microbiol. Biotechnol., 51:407, 1997). However, since this method is based on a single omission technique involving repeated trial and error, it requires a great deal of time, human effort, and is not cost effective. To compensate for the shortcomings of the single omission technique, several statistical techniques have been recently suggested for developing a culture medium, but are not widely used due to a low probability of success associated with these statistical techniques.
After identifying all the nutrient components necessary for microorganism cultivation using a single omission technique or a statistical technique, determination of an optimum amount of each nutrient component to be added to the culture medium, necessary to promote the microorganism growth, is required. This also requires a great deal of time, human efforts, and is not cost effective. In particular, a disadvantage in that a considerable number of microorganisms disadvantageously cannot grow in a synthetic medium containing all amino acids and vitamins has been reported, and a majority of microorganisms grow better in a culture medium containing peptones or yeast extract added, as a nutrient component, to a complex medium rather than a synthetic medium. First of all, since chemically defined nutrient components added to a synthetic medium such as amino acid or vitamin are costly, a limitation exists in using a synthetic medium containing such expensive nutrient components in the production of biofuels and biochemicals on an industrial scale through microorganism cultivation.
In the production of biofuels and biochemicals through microorganism cultivation, a complex medium containing peptones, corn steep liquor, or yeast extract that includes most of all the nutrient components necessary for microorganism cultivation, although the nutrient components are chemically undefined, is being widely used. A peptone is a product obtained in the hydrolysis of protein, and when converted to amino acid by peptidase, can be used as a carbon source in the cultivation of microorganism. However, use of peptones to produce biofuels and biochemicals on an industrial scale, through microorganism cultivation, is too expensive. Corn steep liquor is a by-product derived from corn wet milling, and due to a very low cost, has been widely used as an important nutrient component of a culture medium for microorganism (Liggett et al., Bacteriol. Rev., 12:297, 1948; Liggett et al., Bacteriol. Rev., 12:300, 1948). Also, corn steep liquor is a good carbon source for microorganism cultivation and has an advantage of including other various nutrient components, as well as vitamins and amino acids (Atkinson et al., Biochemical Engineering and Biotechnology Handbook, The Nature Press, NY, 57, 1983; Miller et al., Manual of Industrial Microbiology and Biotechnology, American Society of Microbiology, Washington, D.C., 122, 1986; Akhtar et al., Tapi J. 80:161, 1997). Although corn steep liquor is a suitable, low cost, nutrient component in the cultivation of microorganisms corn steep liquor has a disadvantage of having to be added to a culture medium in larger amounts, when compared to peptones or yeast extract. Also, since corn steep liquor includes an unknown component that inhibits microorganism growth, when compared to the same amount of yeast extract, corn steep liquor is inferior in terms of microorganism growth rate, concentration of microorganisms, and productivity of a target substance (Amartey et al., Bullet. Chem. Technol., Macedonia, 19:65, 2000; Silveira et al., Appl. Microbiol. Biotechnol., 55:442, 2001; Underwood et al., Appl. Environ. Microbiol., 70:2734).
Yeast extract is proved as a superior nutrient component for microorganism cultivation by many researchers, and in particular, yeast extract has been reported to improve the productivity of a target substance through microorganism cultivation by promoting microorganism growth (Laube et al., Biotechnol. Lett., 6:535, 1984; Bibal et al., Appl. Microbiol. Biotechnol., 30:630, 1989; Aeschlimann et al., Appl. Microbiol. Biotechnol., 32:398, 1990; Norton et al., J. Dairy Sci., 77:2494, 1994; Kazamias et al., Appl. Environ. Microbiol., 61:2425, 1995; Potvin et al., J. Microbiol. Methods, 29:153, 1997; Bafrncova et al., Biotechnol. Lett., 21:337, 1999; Bury et al., Czech J. Food Sci., 19:166, 2001). Also, since yeast extract includes a considerable amount of carbohydrates and soluble sugars, yeast extract provides both a nutrient component and a carbon source in the cultivation of microorganisms (Revillion et al., Braz. Arch. Biol. Technol., 46:121, 2003; Ojokoh et al., Afr. J. Biotechnol., 4:1281, 2005). Due to this superior characteristic, yeast extract is being widely used as an additive in the food industry. Generally, yeast extract is made by artificially growing a strain of Saccharomyces cerevisiae that is a species of yeast used in production of beer and bread, followed by autolysis. However, as a matter of fact, yeast extract made through this process is improper as a nutrient component of a culture medium for producing biofuels and biochemicals on an industrial scale because of a very high cost. When producing lactic acid using substantially 2 g/L of yeast extract as a nutrient component, it is reported that the cost of the yeast extract corresponds to 32% of the total production cost of the lactic acid (Mulligan et al., Biotechnol. Bioeng., 38:1173, 1991).
Studies on the production of biofuels and biochemicals through microorganism cultivation are directed to the development of low-priced carbon sources and culture mediums, development of a fermentation process, development of a separation and purification process, and development of a superior strain. Currently, when producing biofuels and biochemicals on an industrial scale through microorganism cultivation, a complex medium containing corn steep liquor, yeast extract, and peptones as major nutrient components is being widely used. As described in the foregoing, corn steep liquor has a low cost but cannot guarantee productivity of a target substance since it inhibits microorganism growth. Yeast extract and peptones are superior nutrient components but cannot guarantee economical efficiency since they are costly. Accordingly, there is an urgent demand for development of a culture medium, of low cost, that sufficiently contains superior nutrient components and can guarantee productivity and economical efficiency in production of biofuels and biochemicals on an industrial scale.
Some studies have been conducted on recycling a by-product produced in the microorganism fermentation. For example, U.S. Pat. No. 4,578,353 discloses a process in which a non-fermented solid carbohydrate residue generated in the alcohol fermentation is hydrolyzed in a sludge tank again and the hydrolysate is re-supplied as a carbon source to the same alcohol fermentation tank. WO 2008/115080 discloses a process in which two alcohol fermentation tanks are linked and an organic acid and a gaseous component produced as a by-product in each fermentation tank are selectively separated and recycled. However, these arts are limited in that they selectively recycle only some components of a material provided as a feedstock or a substance produced as a by-product in a fermentation tank by microorganisms.
Unlike fuels and chemicals produced using fossil resources as a feedstock, biofuels and biochemicals can be obtained using, as a feedstock, biomass that is a biological resource enabling complete circulation of resources. Recently, in an effort to overcome the energy crisis brought about by exhaustion of fossil fuels and environmental crises such as a climatic change caused by a build-up of greenhouse gases in the atmosphere, demand is increasing for carbon-neutral or carbon-zero and environmentally friendly biofuels and biochemicals that enable the complete circulation of resources and carbon dioxide. In particular, since delivery and storage of liquid biofuels such as, ethanol and butanol, is easy and thus, suitable for use as transportation fuels, liquid biofuels are gaining attention.
To date, biofuels and biochemicals have been primarily produced through microorganism fermentation of glucose obtained by hydrolyzing corn starch or wheat, or sucrose included in sugar cane. However, as crop costs continue to increase and use of food resources in production of biofuels and biochemicals is being raised as a moral issue, attempts have been actively made, on a global scale, to efficiently produce biofuels and biochemicals by using, as a feedstock, lignocellulosic biomass since it is an abundant non-food biological resource that is readily available across the world.
Lignocellulosic biomass may be largely divided into a woody biomass and a grassy biomass, and contains cellulose, hemicellulose, and lignin as major components.
After lignocellulosic biomass is removed through physical and chemical pre-processing using an acid, a base, or a vapor, the pre-processed lignocellulosic biomass is hydrolyzed by a hydrolase so that cellulose is converted to hexose including glucose, and hemicellulose is converted to pentose including xylose and arabinose, as well as hexose. It is known that most microorganisms are capable of utilizing hexose, in particular, glucose as a carbon source and an energy source during fermentation, however, some microorganisms are incapable of utilizing pentose as a carbon source and an energy source.
Accordingly, when producing biofuels and biochemicals through fermentation of a microorganism either incapability of or having a remarkably low capability of utilizing pentose, it is impossible to use pentose that is about 35% of the total sugar derived from lignocellulosic biomass. As a result, a subsequent increase of the feedstock cost would make it difficult to ensure the economical efficiency. Also, due to pentose being present in the wastewater produced after fermentation leading to a rapid increase in the chemical oxygen demand and biological oxygen demand, a separate process for treating the wastewater is required.
Accordingly, there is a need for development of a method for preparing biofuels and biochemicals in an economical and environmentally friendly manner while making efficient use of pentose and hexose derived from lignocellulosic biomass.